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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57505—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the blood, e.g. leukaemia
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • G01N2333/91205—Phosphotransferases in general
  • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
  • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/22—Haematology
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7023—(Hyper)proliferation

Definitions

• the present invention relates to methods for assaying JAK2 activity in a red blood cell.
• PV Polycythemia vera
• MPN myeloproliferative neoplasm
• the V617F mutation is detected by a single nucleotide polymorphism genotyping assay performed with DNA extracted from granulocytes. When detected, the percentage of the mutated alleles is quantified by a PCR-based assay using the same DNA.
• the JAK2V617F mutated form may be a prognostic marker because the percentage of circulating mutated alleles (%V617F) appears to be significantly higher in patients who develop vascular complications (8).
• the %V617F is considered as a good tool to monitor minimal residual disease and to evaluate treatment efficacy (9, 10).
• the present invention relates to a method for assaying JAK2 activity in a red blood cell comprising the steps consisting of i) bringing the red blood cell into contact with laminin ii) determining the ability of the red blood cell to adhere to laminin, and iii) concluding that JAK2 is activated when the red blood cell is able to adhere to laminin or concluding that JAK2 is not activated when the red blood cell is not able to adhere to laminin.
• the role of the adhesion molecule Lu/BCAM as a potential biological marker of JAK2V617F activity in PV RBCs was investigated by the inventors in 17 JAK2V617F- positive PV patients.
• the inventors found a significant correlation between the number of adherent RBCs and the percentage of Lu/BCAM-positive RBCs.
• the inventors also demonstrated a strong correlation between RBC adhesion to laminin and the JAK2V617F percentage determined by PCR and represented as the allele burden. This correlation was reinforced in the group of 11 patients with similar percentages of Lu/BCAM-positive RBCs, demonstrating that JAK2V617F is the critical factor controlling the Lu/BCAM activation and adhesion level.
• the Lu/BCAM-mediated adhesion is determined as a biological effect of JAK2V617F activity in PV RBCs.
• the inventors' methods based on a one-hour test, has a clear advantage over the current method used for determining the percentage of the mutated JAK2 allele in the total population of circulating nuclear blood cells and consisting of growing Endogenous Erythroid Colonies (EEC) by performing a two-week cell culture assay.
• EEC Endogenous Erythroid Colonies
• the present invention relates to a method for assaying JAK2 activity in a red blood cell comprising the steps consisting of i) bringing the red blood cell into contact with laminin ii) determining the ability of the red blood cell to adhere to laminin, and iii) concluding that JAK2 is activated when the red blood cell is able to adhere to laminin or concluding that JAK2 is not activated when the red blood cell is not able to adhere to laminin.
• JAK2 refers to a cytoplasmic tyrosine kinase that transduces signals triggered by multiple haemopoietic growth factors such as erythropoietin, in normal and neoplastic cells (1).
• JAK2 activity refers to the kinase activity of JAK2.
• JAK2 activity includes JAK2-mediated Lu/BCAM activation through a Rapl/Akt signaling pathway and activation of other proteins by phosphorylation such as ICAM-4/LW that interacts with integrin ⁇ 3 on the surface of endothelial cells.
• laminin has its general meaning in the art and refers to the major component of the extracellular matrix.
• laminin refers to one of the major functional components of basement membranes, and are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Laminins containing the a4 and a5 chains are the major iso forms found in the vessel wall, with the added contribution of laminin a2 in larger vessels.
• laminin is immobilized onto a solid support.
• solid support refers to a material having a rigid or semi-rigid surface. Such materials will preferably take the form chips, plates, cuvettes, filters, titer plates, beads and the like, that have laminin to the surface of those supports.
• the supports are generally made of conventional materials, e.g., plastic polymers, cellulose, glass, ceramic, stainless steel alloy, and the like.
• the adhesion of the red blood cell to laminin may be determined by any well-known method in the art and typically involves adhesion assays such as described in the prior art (12).
• the adhesion ability is determined in flow condition.
• an assay as described in the EXAMPLE is used for determining the adhesion of the red blood cell to laminin.
• the JAK2 activity may result from a JAK2 gain of function mutation.
• the present invention also relates to a method for determining the presence of a JAK2 gain of function mutation in a red blood cell, comprising the steps consisting of i) bringing the red blood cell into contact with laminin ii) determining ability of the red blood cell to adhere to laminin, and iii) concluding that a JAK2 gain of function mutation is present in the red blood cell when the red blood cell is able to adhere to laminin or concluding that a JAK2 gain of function mutation is absent when the red blood cell is not able to adhere to laminin.
• the activation is not reached by adding to the red blood cell any agent capable of activating JAK2. The activation thus results only from the JAK2 gain of function mutation.
• JAK2 gain of function mutation refers to any mutation in JAK2 which has for consequence that the kinase activity is auto constitutive.
• the gain of function mutation can be a deletion, addition, or substitution of amino acids in the protein, which gives rise to the change in the function of the JAK2.
• the mutation is a somatic mutation.
• a JAK2 gain of function mutation is the somatic JAK2V617F mutation described in James et al. (James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434: 1144-1148).
• JAK2V617F is a point mutation (1849 G for T) in exon 14, causes the substitution of phenylalanine for valine at codon 617 in the JAK homology JH2 domain.
• Other JAK2 gain of function mutations include but are not limited to F537-K539delinsL, H538QK539L, K539L, N542-E543del, E543-D544del, R541-E543delinsK, 1540- E543delinsMK and V617I.
• the present invention also relates to a method for assaying the amount of constitutively active erythroid JAK2 (CAEJ) in a population of red blood cells due to a JAK2 gain of function mutation, comprising the steps consisting of i) bringing the population of red blood cells into contact with laminin and ii) determining the rate of red blood cells which adhere to laminin, wherein said rate is indicative of the amount of constitutively active erythroid JAK2 (CAEJ) in a population of red blood cells due to a JAK2 gain of function mutation.
• CAEJ constitutively active erythroid JAK2
• the expression "amount of constitutively active erythroid JAK2" or “CAEJ amount” refers to the number of red blood cells comprising JAK2 with gain of function mutations; this number is shown by the inventors to reflect the allele burden of a JAK2 gain of function mutation.
• the expression “CAEJ amount” also reflects the percentage of the mutated JAK2 allele in the total population of circulating nucleated blood cells. Typically, the rate is the number of adherent cells per surface unit (e.g. number of cells per mm 2 ).
• the red blood cells may result from a blood sample derived from a patient suffering from a myeloproliferative neoplasm (MPN).
• MPN myeloproliferative neoplasm
• myeloproliferative neoplasm refers to myeloproliferative neoplasm such as revised in the World Health Organisation Classification D47.1.
• the term encompasses a number of entities characterized by uncontrolled marrow proliferation in the presence of intact cellular differentiation and includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).
• PV polycythemia vera
• ET essential thrombocythemia
• PMF primary myelofibrosis
• the methods of the invention may find very different applications.
• the method for assaying the CAEJ amount may be particularly suitable for the prediction of a secondary event in patients suffering from a myeloproliferative neoplasm (MPN).
• the present invention also relates to a method for determining the risk of a secondary event in a patient suffering from a myeloproliferative neoplasm comprising the steps consisting of i) determining the CAEJ amount in a sample of red blood cells obtained from the patient by performing the method as above described ii) comparing the CAEJ amount determined in step i) with a reference value and iii) concluding that the patient has a high risk of having a secondary event when the CAEJ amount determined at step i) is higher than the reference value, or concluding that the patient has a low risk of having a secondary event when the CAEJ amount determined at step i) is lower than the reference value.
• the secondary event is selected from the group consisting of vascular complications, secondary myelofibrosis, pruritus and transformations into leukemia vascular complication.
• vascular complication has its general meaning in the art and includes arterial and venous thrombosis.
• reference value may be determined by carrying out a method comprising the steps of:
• step c) classifying said samples of red blood cells in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples of red blood cells that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples of red blood cells that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples of red blood cells are obtained for the said specific quantification value, wherein the samples of red blood cells of each group are separately enumerated;
• the CAEJ amount has been assessed for 100 samples of red blood cells of 100 patients.
• the 100 samples are ranked according to CAEJ amount.
• Sample 1 has the highest CAEJ amount and sample 100 has the lowest CAEJ amount.
• a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
• the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
• the p value between both subsets was calculated.
• the reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest.
• the CAEJ amount corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of CAEJ amount.
• a single "cut-off value thus allows discrimination between responder or non responder.
• high statistical significance values e.g. low P values
• high statistical significance values e.g. low P values
• a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided.
• minimum threshold of significance e.g. maximal threshold P value
• This range of quantification values includes a "cut-off value as described above. For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. Therefore, a patient may be assessed by comparing values obtained by measuring the CAEJ amount, where values greater than 5 reveal that the patient has a high risk of having a secondary event and values less than 5 reveal that the patient has low risk of having a secondary event.
• a patient may be assessed by comparing values obtained by measuring the CAEJ amount and comparing the values on a scale, where values above the range of 4-6 indicate that the patient has a high risk of having a secondary event and values below the range of 4-6 indicate that the patient has a low risk of having a secondary event, with values falling within the range of 4-6 indicating an intermediate risk.
• the methods of the invention may be also suitable for determining the presence or absence of the gain of function mutation in the erythroid lineage of a patient suffering from a myeloproliferative neoplasm. Determining the presence or absence of constitutively active JAK2 in the erythroid lineage has an impact on patient characterization within the three subpopulations of MPN patients: PV, ET or PMF. In addition, the presence of active JAK2 in the erythroid lineage of ET or PMF patients might give indications about their potential evolution into PV.
• the inventors' methods based on a one-hour test, has a clear advantage over the current method used for the same purpose and consisting of growing Endogenous Erythroid Colonies (EEC) by performing a two-week cell culture assay.
• EEC Endogenous Erythroid Colonies
• the methods of the invention may be also suitable to define a subgroup of patients who are eligible for a treatment with a JAK2 inhibitor. Indeed patient with a high CAEJ amount may be administered with a JAK2 inhibitor.
• JAK2 inhibitors are well known in the art (Tibes R, Bogenberger JM, Geyer HL, Mesa RA. JAK2 inhibitors in the treatment of myeloproliferative neoplasms. Expert Opin Investig Drugs. 2012 Dec;21(12): 1755-74; Dymock BW, See CS. Inhibitors of JAK2 and JAK3: an update on the patent literature 2010 - 2012. Expert Opin Ther Pat.
• the methods of the invention are also particularly suitable for monitoring a treatment of patient suffering from a myeloproliferative neoplasm (e.g. with a JAK2 inhibitor). Typically a decrease in the CAEJ amount indicates that the treatment is effective in the patient.
• the methods of the invention may be also particularly suitable for the in cellulo screening of JAK2 inhibitors.
• said method comprises the steps consisting of i) bringing a red blood cell harbouring a JAK2 gain of function mutation into contact with a candidate compound and ii) determining the ability of the red blood cell to adhere to laminin, wherein a decrease or an absence of adhesion indicate that the candidate compound is an effective in cellulo JAK2 inhibitor.
• PV RBC adhesion is weakly correlated with the percentage of Lu/BCAM-positive RBCs but not with Lu/BCAM MFI values.
• RBC adhesion at 3 dyn/cm 2 as a function of the Lu/BCAM expression level on the surface of RBCs from 17 PV patients, represented as the percentage of circulating RBCs expressing Lu/BCAM (A) and Lu/BCAM MFI (B).
• FIG. 1 PV RBC adhesion as a function of the JAK2V617F allele burden.
• Red blood cell adhesion to laminin 511/521 was measured under flow conditions using Vena8 Endothelial+TM biochips (internal channel dimensions: length 20 mm, width 0.8 mm, height 0.12 mm) and MinisTM Nanopump (Cellix Ltd, Dublin, Ireland), as described (12).
• Laminin 511/521 from human placenta was immobilized on the internal surface of the biochips (1 ⁇ / ⁇ 2 ) at 4°C overnight.
• RBCs were suspended in Hanks balanced salt solution, without calcium chloride and magnesium sulfate (Sigma- Aldrich), supplemented with 0.4% BSA at hematocrit 0.5% and perfused through the biochip channels for 10 min at a shear stress of 0.2 dyn/cm 2 .
• Five minutes washouts with the Hanks/0.4% BSA buffer were performed at 1, 2, 3, 4 and 5 dyn/cm 2 .
• adherent RBCs were counted in 11 representative areas along the centerline of each channel using the AxioObserver Zl microscope and Axio Vision 4 analysis software (Carl Zeiss, Le Pecq, France). Images of the same 11 areas were obtained throughout each experiment using the "Mark and Find" module of Axio Vision analysis software.
• Lu/BCAM Cell surface expression of Lu/BCAM was analyzed using anti-Lu/BCAM F241 mouse mAb followed by a PE-conjugated secondary anti-mouse IgG antibody, and a BD FACScanto II flow cytometer (Becton-Dickinson, San Jose, CA) with FACSDiva software (v6.1.2) for acquisition and analysis.
• PV RBC adhesion to laminin is variable and weakly correlated with Lu/BCAM expression
• Lu/BCAM phosphorylation by a JAK2V617F/Rapl/Akt pathway (12).
• the inventors performed adhesion assays with blood samples from 17 JAK2V617F-positive PV patients and determined the number of RBCs adhering to laminin at 3 dyn/cm 2 .
• the adhesion values were variable, ranging from 14 to 1021 RBC/mm 2 (Table 1). Because Lu/BCAM erythroid expression is heterogeneous, with a variable surface expression level among the population and a percentage of Lu/BCAM-negative RBCs within the same individual, the inventors asked whether PV RBC adhesion to laminin could be conditioned by Lu/BCAM expression pattern.
• the inventors performed flow cytometry analyses to characterize Lu/BCAM expression in all blood samples.
• the percentage of Lu/BCAM-positive RBCs ranged from 38 to 89.1, and the expression level, estimated by the mean fluorescence intensity (MFI), from 1249 to 3464 (Table 1).
• MFI mean fluorescence intensity
• the number of adherent RBCs was plotted against the percentage of Lu/BCAM-positive RBCs or the MFI of each blood sample.
• PV RBC adhesion to laminin reflects the JAK2V617F allele burden
• the V617F mutation is detected in DNA samples extracted from patients' granulocytes. The mutation is thus detected in myelocytes but not in cells from the erythroid lineage.
• quantifying a mutation by a PCR-based method does not reflect the mutated protein levels.
• the inventors revealed a biological marker that allows estimating the V617F mutational load at the protein level in PV RBCs. Inventor's assay does not quantify JAK2V617F mere expression but measures the functional level of this enzyme.
• the Lu/BCAM-mediated adhesion is determined as a direct biological effect of JAK2V617F activity in PV RBCs.
• This assay is interesting in other MPNs with partial JAK2V617F erythroid occurrence, such as Essential Thrombocythemia (ET), and primary myelofibrosis (PMF) as it discriminates between JAK2V617F-positive and JAK2V617F-negative RBC samples and thus give an indication about the presence or absence of this mutation in the erythroid lineage of these patients.
• JAK2V617F erythroid occurrence such as Essential Thrombocythemia (ET), and primary myelofibrosis (PMF)
• Table 1 The list of the JAK2V617F-positive PV patients included in this invention, numbered from 1 to 17, indicating their adhesion level (RBCs/mm 2 ), their relative values of Lu/BCAM expression (% and MFI) and of JAK2V617F allele burden (%JAK2V617F).
• V617F A unique activating mutation in JAK2 (V617F) is at the origin of polycythemia vera and allows a new classification of myeloproliferative diseases. Hematology Am Soc Hematol Educ Program. 2005: 195-200.
• Wautier MP El Nemer W, Gane P, et al. Increased adhesion to endothelial cells of erythrocytes from patients with polycythemia vera is mediated by laminin alpha5 chain and Lu/BCAM. Blood. 2007;110(3):894-901.

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